Antioxidant and Anti-inammatory Assays Conrm Bioactive

Compounds in Ajwa Date Fruit

Chuan-Rui Zhang,

Saleh A. Aldosari,

Polana S. P. V. Vidyasagar,

Karun M. Nair,

and Muraleedharan G. Nair*

,

Bioactive Natural Products and Phytoceuticals Laboratory, Department of Horticulture, Michigan State University, East Lansing,
Michigan 48824, United States

College of Food and Agriculture Sciences, Chair of Date Palm Research, King Saud University, Riyadh 11451, Saudi Arabia
ABSTRACT: Ajwa, a variety of date palm Phoenix dactylifera L., produces the most expensive date fruits. Percentages of seed,
moisture, fructose, glucose, soluble protein, and ber in Ajwa dates were 13.24, 6.21, 39.06, 26.35, 1.33, and 11.01, respectively.
The ethyl acetate, methanolic, and water extracts of Ajwa dates, active at 250 g/mL in the MTT assay, inhibited lipid
peroxidation (LPO) by 88, 70, and 91% at 250 g/mL and cyclooxygenase enzymes COX-1 by 30, 31, and 32% and COX-2 by
59, 48, and 45% at 100 g/mL, respectively. Bioactivity-guided purications aorded compounds 17, in addition to phthalates
and fatty acids. Compounds 13 showed activity at 100 g/mL in the MTT assay; inhibited COX-1 enzyme by 59, 48, amd 50%
and COX-2 enzyme by 60, 40, amd 39% at 50 g/mL; and inhibited LPO by 95, 58, amd 66% at 100 g/mL, respectively. The
soluble protein fraction was also very active in both antioxidant and anti-inammatory assays.
KEYWORDS: sugars, avonoid glycosides, triterpenoids, triglycerides, steroids, phthalates, fatty acids

INTRODUCTION
Numerous varieties of date palm, Phoenix dactylifera L.
(Palmaceae), are grown in the Middle East, North Africa,
South Asia, and the United States. Major date palm varieties
grown in the United States are Deglet Noor and Medjool. Ajwa
date fruits, soft and dry, are from a date palm variety cultivated
in the Al Madinah region of western Saudi Arabia. This date
variety is ascribed as having great medicinal value. Reference
about Ajwa dates was made in Hadith and Islamic historical
literature because it is believed that eating this variety will cure
many chronic ailments. The Ajwa date fruit is one of the most
popular and expensive dates, fetching 3 times the price of the
next best variety, and belongs to the holy city of Al Madinah Al
Munawara and its adjoining areas in Saudi Arabia.
Date fruit is consumed as a staple food or as an important
component of the diet in the Middle East region. This fruit is
considered to be highly nutritional because of its rich sugar con-
tent in the form of fructose and glucose, dietary ber, vitamins,
and minerals.
1,2
An overall composition including functional
and nutritional quality of a variety of date palm fruits has been
also reported.
3,4
For example, the aqueous extract of date fruit
showed antioxidant and antimutagenic activities, which was
attributed to the presence of compounds with free radical
scavenging activity.
5
Several varieties of date fruits from Saudi
Arabia and Algeria showed antioxidant activity due to their
phenolic content.
69
The carotenoid prole of some Algerian
date fruit varieties has also been investigated.
10
The avonoid
glycoside and procyanidin composition of Deglet Noor dates
from California was determined using liquid chromatography
electrospray ionizationtandem mass spectrometry (LC-ESI/
MS/MS).
11
A recent paper described the chemical constituents
and biological activity of date seeds.
12
Although limited nutri-
tional, chemical, and bioactivity studies are available on date
fruits, this is the rst attempt of a bioassay-guided evaluation of
constituents in dates and characterization of pure and active
isolates from it.
In this study, antioxidant and anti-inammatory activities of
hexane, ethyl acetate, methanolic, and water extracts of Ajwa
date fruits were determined using 3-(4,5-dimethylthiazole-2-yl)-
2,5-diphenyltetrazolium bromide (MTT),
1315
lipid peroxida-
tion (LPO),
1418
and cyclooxygenase enzymes (COX-1 and -2)
inhibitory
1418
assays as per published studies from our labo-
ratory. Also, we report the purication, structure elucidation,
and bioactivity studies of pure isolates as a measure to deter-
mine their functional food quality.

MATERIALS AND METHODS

Safety. There are no safety concerns.
General Experimental Procedures. All solvents used for
isolation and purication were of ACS reagent grade (Sigma-Aldrich
Chemical Co., St. Louis, MO, USA). Merck silica gel (60 mesh size,
3570 m) with particle size of 60 m was used for preparative
medium-pressure liquid chromatography (MPLC). Silica gel plates
(250 and 500 m; Analtech, Inc., Newark, DE, USA) were used for
preparative thin-layer chromatography (TLC). TLC plates were
viewed under UV light at 254 and 366 nm in a Spectroline CX-20
ultraviolet uorescence analysis cabinet (Spectroline Corp., Westbury,
NY, USA) and sprayed with 10% sulfuric acid solution. NMR spectra
were recorded on a 500 MHz (Varian Unity 500,
1
H NMR) and 125
MHz (Varian Unity 500,
13
C NMR) VRX instruments. The mass
spectrum was recorded on a Waters Quattro micro API LC/MS/MS
spectrometer. 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), tert-butylhydroquinone (TBHQ), butylated hydrox-
yanisole (BHA), butylated hydroxytoluene (BHT), aspirin, naproxen,
Received: March 27, 2013
Revised: May 28, 2013
Accepted: May 28, 2013
Published: May 28, 2013
Article
pubs.acs.org/JAFC
2013 American Chemical Society 5834 dx.doi.org/10.1021/jf401371v | J. Agric. Food Chem. 2013, 61, 58345840
and ibuprofen were purchased from Sigma-Aldrich Chemical Co.
Similarly, the nonsteroidal anti-inammatory drug (NSAIDs) Celebrex
was a physicians professional sample provided by Dr. Subhash Gupta,
Sparrow Pain Center, Sparrow Hospital, Lansing, MI, USA. COX-1
and -2 enzymes were prepared in our laboratory from ram seminal
vesicles (Oxford Biomedical Research, Inc., Oxford, MI, USA) and
insect cells cloned with human PGHS-2 enzyme, respectively.
Arachidonic acid was purchased from Oxford Biomedical Research,
Inc. 1-Stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine (SLPC) was
purchased from Avanti Polar Lipids (Alabaster, AL, USA). The
uorescent probe 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic
acid was purchased from Molecular Probes (Eugene, OR, USA). All
enzymes and reagents were stored in the Bioactive Natural Products
and Phytoceuticals Laboratory at Michigan State University (East
Lansing, MI, USA). MTT antioxidant activity was tested on a Bio-Tek
Elx800 universal microplate reader (Bio-Tek Instruments, Inc.,
Winooski, VT, USA). COX assays were performed in an Instech
micro oxygen chamber and electrode (Instech Laboratories, Plymouth
Meeting, PA, USA) attached to a YSI model 5300 biological oxygen
monitor (Yellow Springs Instrument, Inc., Yellow Springs, OH, USA).
LPO assay was tested on a Turner model 450 uorometer (Barnstead/
Thermolyne Corp., Dubuque, IA, USA).
Extraction and Isolation. Ajwa fruit samples were procured from
a farm located in the Madinah region. This farm cultivates only Ajwa
date palms. From ve palms, 1 kg of fruits was drawn and mixed in a
bag. From this, 1 kg aliquots of fruit sample were packed in cardboard
boxes and sent to Riyadh for onward shipment to Michigan State
University. At the time of harvest, the fruit was in Tamar or ripened
stage, and identication was based on the local knowledge of the
farmer and as per information provided by the Ministry of Agriculture,
Saudi Arabia.
19
Ajwa dates (740 g) were pitted to remove seeds (98 g). The pit-free
fruit (642 g) was blended with 1.5 L of water and the puree lyophilized
to yield powdered fruit (596 g). The dry powder (596 g) was packed
in a glass column and eluted sequentially with hexane (2 L), ethyl
acetate (2 L), and methanol (2 L). The evaporation of organic sol-
vents under vacuum at 35 C aorded hexane (40 mg), ethyl acetate
(480 mg), and methanolic extracts (348.5 g), respectively. The residue
left in the column was removed, blended with 2 L of water,
centrifuged, and lyophilized (supernatant and residue separately) to
aord water-soluble (170 g) and -insoluble (brous, 77 g) fractions.
The hexane extract was combined with the ethyl acetate extract on
the basis of TLC proles. An aliquot of this combined extract (500 mg)
was fractionated by silica gel vacuum liquid chromatography (VLC) and
eluted under gradient conditions using hexane/acetone (10:1, 3:1, 1:1,
v/v), followed by CHCl
3
/MeOH (10:1, 3:1, 1:1, v/v). The fractions
collected were A, 126 mg; B, 85 mg; C, 44 mg; D, 60 mg; and E, 185
mg. An aliquot of fraction A (115 mg) was puried by preparative TLC
(hexane/acetone, 15:1, v/v; and CHCl
3
/MeOH, 200:1, v/v) to yield
compounds 2 (10.7 mg), 3 (5 mg), 4 (3.6 mg), and 7 (4.7 mg), bis(2-
ethylhexyl) terephthalate (3 mg), and bis(2-ethylheptyl) phthalate (7 mg).
An aliquot of fractopm B (68 mg) was puried by preparative TLC
(hexane/acetone, 10:1; and CHCl
3
/MeOH, 100:1) to aord a fatty
acid mixture containing equal amounts of linoleic, oleic, and stearic
acids (21.5 mg). An aliquot of fraction C (30 mg) was puried by
preparative TLC (CHCl
3
/MeOH, 15:1) to yield 5 (8 mg). Similarly,
fraction D (20 mg) was puried by preparative TLC (CHCl
3
/MeOH,
10:1) to aord 6 (6.4 mg).
An aliquot of the methanolic extract (25 g) was fractionated by
MPLC (C18 column) and eluted with MeOH/H
2
O (gradient elution,
1:9, 3:7, 5:5, 7:3, 9:1, v/v) and nally with MeOH (100%) to yield
fractions F, 24.8 g; G, 95 mg; and H, 101 mg. Major compounds
in fraction F were determined as -D-glucopyranose (22.9%), -D-
glucopyranose (16.7%), -D-fructopyranose (45.3%), and -D-
fructofuranose (13.4%) on the basis of their NMR spectral data and
TLC proles with authentic samples. Compound 1 (1.8 mg) was
isolated from fraction G by preparative TLC (CHCl
3
/MeOH/H
2
O,
4:1:0.1, v/v). Fraction H was identical to the ethyl acetate extract
based on TLC and hence was not puried further.
Tituration of a portion of the water extract (5 g) with MeOH
(50 mL 3) yielded MeOH-soluble (I, 4.58 g) and -insoluble (J, 421 mg)
fractions. Analysis of fraction I revealed that it contained sugars
(99.5%), primarily fructose and glucose, on the basis of TLC proles
with authentic samples of fructose and glucose. Insoluble fraction J was
dissolved in water (15 mL) to yield water-soluble fraction (K, 289 mg)
and residue (L, 132 mg). Preliminary NMR study suggested that K
was proteinaceous in nature. This fraction was not studied further
because the scope of it was beyond this study.
Chrysoeriol-7-O-(2,6-dirhamnosyl)-glucoside 1: yellow powder;
1
H NMR (500 MHz, CD
3
OD) 7.55 (1H, dd, J = 9.0, 2.0 Hz, H-6),
7.51 (1H, d, J = 2.0 Hz, H-2), 6.93 (1H, d, J = 9.0 Hz, H-5), 6.83
(1H, d, J = 2.0 Hz, H-8), 6.69 (1H, s, H-3), 6.47 (1H, d, J = 2.0 Hz,
H-6), 5.28 (1H, J = 1.5 Hz, H-1), 5.20 (1H, d, J = 8.0 Hz, H-1),
4.57 (1H, br s, H-1), 3.96 (3H, s, H-7), 3.303.95 (14H, m, H-2,
3, 4, 5, 6, 2, 3, 4, 5, 2, 3, 4, 5), 1.32 (6H, d, J = 5.5
Hz, H-6, 6).
20
Lup-20(29)-en-3-one 2: white powder;
1
H NMR (500 MHz, CDCl
3
)
4.70 (1H, br d, J = 2.0 Hz, H-29a), 4.58 (1H, br t, J = 2.0 Hz,
H-29b), 2.352.55 (3H, m, H
2
-2 and H-19), 1.851.95 (2H, m, H-1a
and H-21a), 1.69 (3H, s, H
3
-30), 1.08 (6H, s, H
3
-23 and H
3
-26), 1.04
(3H, s, H
3
-24), 0.97 (3H, s, H
3
-27), 0.94 (3H, s, H
3
-25), 0.81 (3H, s,
H
3
-28).
21,22
Lupeol 3: white powder;
1
H NMR (500 MHz, CDCl
3
) 4.70 (1H,
br d, J = 2.0 Hz, H-29a), 4.58 (1H, br dd, J = 1.5, 1.0 Hz, H-29b), 3.19
(1H, dd, J = 11.5, 5.0 Hz, H-3), 2.38 (1H, m, H-19), 1.95 (2H, m,
H
2
-21), 1.69, 1.04, 0.98, 0.95, 0.84, 0.80, 0.77 (each 3H, s, CH
3
7).
23
1,2-Dilinoleoyl-3-stearin 4: colorless oil; APCI-MS, m/z 883 [M +
H]
+
;
1
H NMR (500 MHz, CDCl
3
) 5.35.4 (8H, m, 9, 10, 12,
13, 9, 10, 12, 13), 5.27 (1H, m, H-2), 4.30 (2H, dd, J = 12.0, 4.3
Hz, H-1a, 3a), 4.15 (2H, dd, J = 12.0, 6.0 Hz, H-1b, 3b), 2.78 (4H, m,
H
2
-11, 11), 2.32 (6H, m, H
2
-2, 2, 2), 2.05 (8H, m, H
2
-8, 14,
8, 14), 1.62 (6H, m, H
2
-3, 3, 3), 1.21.4 (56H, m, H
2
-4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 4, 5, 6, 7, 15, 16,
17, 4, 5, 6, 7, 15, 16, 17), 0.89 (9H, m, Me-18, 18, 18);
13
C NMR (125 MHz, CDCl
3
) 173.2, 172.8 (C-1, 1, 1), 130.2,
130.0, 128.0, 127.8 (C-9, 10, 12, 13, 9, 10, 12, 13), 68.9 (C-
1), 62.1 (C-2, 3), 34.2, 34.0, (C-2, 2, 2), 31.9, 31.5 (C-16, 16,
16), 29.029.7 (C-4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
4, 5, 6, 7, 15, 16, 4, 5, 6, 7, 15, 16), 27.2, 27.1 (C-8,
14, 8, 14), 25.6 (C-11, 11), 24.8 (C-3, 3, 3), 22.6, 22.5
(C-17, 17, 17), 14.1, 14.0 (C-18, 18, 18).
24
-Sitosteryl-3-glucopyranoside-6-O-palmitate 5: colorless oil;
1
H NMR (500 MHz, CDCl
3
) 5.37 (1H, m, H-6), 4.50 (1H, dd, J =
11.7, 4.5 Hz, H-6a), 4.39 (1H, d, J = 7.4 Hz, H-1), 4.27 (1H, br d, J =
11.7 Hz, H-6b), 3.33.7 (5H, m, H-3, 2, 3, 4, 5), 1.02 (3H, s, Me-
19), 0.93 (3H, d, J = 6.3 Hz, Me-21), 0.89 (3H, t, J = 7.1 Hz, Me-16),
0.85 (3H, t, J = 7.5 Hz, Me-29), 0.84 (3H, d, J = 6.8 Hz, Me-27), 0.82
(3H, d, J = 6.8 Hz, Me-26), 0.69 (3H, s, Me-18);
13
C NMR (125
MHz, CDCl
3
) 174.7 (C-1), 140.3 (C-5), 122.2 (C-6), 101.2 (C-1),
79.6 (C-3), 76.0 (C-3), 74.0 (C-5), 73.6 (C-2), 70.1 (C-4), 63.5
(C-6), 56.8 (C-14), 56.1 (C-17), 50.2 (C-9), 45.9 (C-24), 42.3
(C-13), 39.8 (C-12), 38.9 (C-4), 37.3 (C-1), 36.7 (C-10), 36.2
(C-20), 34.2 (C-2), 34.0 (C-22), 31.9 (C-7, 8, 14), 29.229.7 (C-2,
413), 28.3 (C-16), 26.1 (C-23), 25.0 (C-3), 24.3 (C-15), 23.1
(C-28), 22.7 (C-15), 21.1 (C-11), 19.8 (C-27), 19.4 (C-19), 19.0
(C-26), 18.8 (C-21), 14.1 (C-16), 12.0 (C-29), 11.9 (C-18).
25
Bis-(2-ethylhexyl) terephthalate (PH1): colorless oil; APCI-MS,
m/z 391 [M + H]
+
;
1
H NMR (500 MHz, CDCl
3
) 8.11 (4H, s, H-3,
4, 6, 7), 4.28 (4H, m, H
2
-1, 1), 1.75 (2H, m, H-2, 2), 1.301.50
(16H, m, H
2
-3, 3, 4, 4, 5, 5, 7, 7), 0.96 (6H, t, J = 7.5 Hz, H
3
-8,
8), 0.92 (6H, t, J = 7.1 Hz, H
3
-6, 6);
13
C NMR (125 MHz, CDCl
3
)
165.9 (C-1, 8), 134.2 (C-2, 5), 129.4 (C-3, 4, 6, 7), 67.7 (C-1, 1),
38.9 (C-2, 2), 30.5 (C-3, 3), 28.9 (C-4, 4), 23.9 (C-7, 7), 22.9
(C-5, 5), 14.0 (C-6, 6), 11.0 (C-8, 8).
28
Bis(2-ethylheptyl) phthalate (PH2): colorless oil; APCI-MS, m/z
419 [M + H]
+
;
1
H NMR (500 MHz, CDCl
3
) 7.71 (2H, br dd, J =
8.5, 3.5 Hz, H-3, 5), 7.54 (2H, br dd, J = 8.5, 3.5 Hz, H-4, 6), 4.22
(4H, m, H
2
-1, 1), 1.69 (2H, m, H-2, 2), 1.251.46 (20H, m, H
2
-3,
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401371v | J. Agric. Food Chem. 2013, 61, 58345840 5835
3, 4, 4, 5, 5, 6, 6, 8, 8), 0.93 (6H, m, H
3
-9, 9), 0.89 (6H, m,
H
3
-7, 7).
29
-D-Fructopyranose and -D-fructofuranose mixture: white
powder;
1
H NMR (500 MHz, D
2
O) 4.16 (m, fur H-3, 4), 4.09
(m, pyr H-5, 6a), 3.95 (m, pyr H-4), 3.88 (m, fur H-5, 6a), 3.84 (m,
pyr H-3), 3.76 (m, pyr H-1a, 6b), 3.72 (m, fur H-6b), 3.64 (m, fur
H-1a), 3.61 (m, pyr H-1b), 3.60 (m, fur H-1b);
13
C NMR (125 MHz,
D
2
O) 103.0 (fur C-2), 99.6 (pyr C-2), 82.2 (fur C-5), 76.9 (fur C-3),
76.0 (fur C-4), 71.2 (pyr C-4), 70.7 (pyr C-5), 69.1 (pyr C-3), 65.4
(pyr C-1), 64.9 (pyr C-6), 64.2 (fur C-1), 63.8 (fur C-6).
31
-D-Glucopyranose and -D-glucopyranose mixture: white
powder;
1
H NMR (500 MHz, D
2
O) 5.28 (m, H-1), 4.69 (m,
H-1), 3.863.94 (m, H-4, 6a, H-4, 6a), 3.743.84 (m, H-6b,
H-3, 6b), 3.423.60 (m, H-3, 5, H-2, 5), 3.29 (m, H-2);
13
C
NMR (125 MHz, D
2
O) 97.4 ( C-1), 93.6 ( C-1), 77.4 ( C-5),
77.3 ( C-3), 75.6 ( C-2), 74.3 ( C-3), 73.0 ( C-5), 72.9 ( C-2),
71.2 ( C-4), 71.1 ( C-4), 62.3 ( C-6), 62.1 ( C-6).
31
Proteinaceous fraction K: brown powder;
1
H NMR (500 MHz,
D
2
O) 5.70 (m), 5.11 (m), 3.004.60 (m), 2.61 (m), 2.46 (m), 2.33
(m), 2.06 (m);
13
C NMR (125 MHz, D
2
O) 170184, 94110, 63
82, 55.5, 46.8, 44.5. The overlapping signals in
1
H and
13
C NMR
spectra and the chemical shifts suggested that fraction K was protein-
aceous in nature. The exact nature and structure determination of it
requires additional purication and detailed high-resolution NMR and
mass spectral analyses of the resulting fractions or isolates. Because
characterization of proteins was not within the objectives of this
research, it was kept aside for future investigations.
MTT Antioxidant Assay. The MTT assay was performed
according to our previous paper.
1315
Stock solutions of test extracts,
selected fraction K and compounds, and positive controls (vitamin C
and TBHQ) were prepared in DMSO (10 mg/mL for extracts, 2 mg/mL
for fraction, 4 mg/mL for compounds 13, and 1 mg/mL for controls,
compound 4, phthalates, and mixture of fatty acids). An aliquot of 10 L
of test samples, 190 L of MTT water solution (1 mg/mL), and 200 L
of DMSO was vortexed in a capped glass vial (2 mL) for 1 min, which
was then incubated at 37 C for 24 h. An aliquot (200 L) of the reaction
mixture was pipetted to a 96-well cell culture plate, and the absorbance
was tested at 570 nm in duplicate on a Bio-Tek Elx800 universal
microplate reader (Bio-Tek Instruments, Inc.). The assay was
conducted in duplicate and repeated twice.
Lipid Peroxidation Inhibitory Assay. The extract (250 g/mL),
fraction K (50 g/mL), compounds 13 (100 g/mL), compound 4,
phthalates and mixture of fatty acids (25 g/mL), and positive
controls (BHA, BHT, and TBHQ at 10 M) were tested for lipid
peroxidation (LPO) inhibitory activities by using uorescence
spectroscopy on a Turner model 450 uorometer (Barnstead/
Thermolyne Corp.) according to the reported procedure.
1418
The
liposome, unilamellar vesicles (ULV), was prepared according to the
published procedure. The peroxidation was initiated by the addition
of 20 L of FeCl
2
4H
2
O (0.5 mM) to the assay mixture [HEPES
(100 L), 1 M NaCl (200 L), N
2
-sparged Millipore water (1.64 mL),
DMSO or test sample (20 L)] and 20 L of liposome suspension.
The uorescence was monitored at 0, 1, and 3 min and every 3 min
thereafter up to 21 min. The decrease in uorescence intensity over
time (21 min) indicated the rate of peroxidation. Each sample was
assayed in duplicate, and the percent inhibition was calculated with
respect to DMSO control.
COX-1 and -2 Enzyme Inhibitory Assay. The COX-1 and -2
enzyme (prepared from ram seminal vesicles and insect cells cloned
with human COX-2 enzyme, respectively, in our laboratory) inhibitory
eects of test samples were measured by monitoring the initial rate of
O
2
uptake using an Instech micro oxygen chamber and electrode
(Instech Laboratories) attached to a YSI model 5300 biological oxygen
monitor (Yellow Springs Instrument, Inc.) at 37 C following the
published procedure.
1418
The test samples (6 L) were initially
added to the chamber full of assay buer (Tris 1 mM phenol buer,
600 L, pH 7) and hemoglobin (17 g). COX-1 or COX-2 enzyme
(20 L) was then added and incubated for 2 min. Arachidonic acid
(10 L of solution at 1 mg/mL) was added to initiate the reaction.
The data were recorded using QuickLog for Windows data acquisition
and control software (Strawberry Tree Inc., Sunnyvale, CA, USA).
The extract and compounds were tested at 100 and 50 g/mL
concentrations, respectively. The positive controls, commercial aspirin,
Celebrex, naproxen, and ibuprofen were tested at 108, 1, 15, and
12 g/mL, respectively. Each sample was tested in duplicate, and the
percent inhibition calculated with respect to DMSO control.
Figure 1. (A) Absorbance values at 570 nm of extracts at 250 g/mL
and proteinaceous fraction K at 50 g/mL obtained after reaction with
MTT at 37 C. Vitamin C and TBHQ were used as positive controls
at 25 g/mL. (B) Inhibition of LPO by extracts at 250 g/mL and K
at 50 g/mL. Commercial antioxidants BHA, BHT, and TBHQ were
tested at 10 M. The oxidation of lipid was initiated by the addition of
Fe
2+
ions. (C) COX-1 and COX-2 enzyme inhibitory activities of
extracts at 100 g/mL, K at 50 g/mL, and commercial NSAIDs
aspirin, Celebrex, naproxen, and ibuprofen used as positive control at
108, 1, 15, and 12 g/mL, respectively. The standard error of the
mean was represented for n = 4 (P < 0.05, t test, paired, two tailed).
For the COX assay, vertical bars represent the standard deviation of
each data point (n = 2). The varying concentrations of positive
controls used in these assays were to yield comparable activity proles
between 0 and 100% by test extracts, fraction, and positive controls
alike.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401371v | J. Agric. Food Chem. 2013, 61, 58345840 5836

RESULTS AND DISCUSSION

The Ajwa dates used in this study was deep-brown in color and
nonsticky compared to many varieties of dates available to con-
sumers. To determine the accurate dry weight of the dates used
for extraction, the pit-free fruits were blended with reverse osmosis
(RO) water and lyophilized. The lyophilized date powder was
then sequentially extracted with hexane, ethyl acetate, methanol,
and water to aord lipid-soluble compounds (e.g., pigments,
triglycerides, fatty alcohols, and acids), lipid-insoluble com-
pounds (e.g., phenolics, its glycosides, and steroidal glycosides),
and water-soluble compounds (e.g., sugars, glycosides,
polysaccharides, and proteins). The yield of hexane extract
was minute in quantity and combined on the basis of the TLC
proles prior to purication. The bulk of soluble compounds
in Ajwa date fruit was sugars, the main constituent in both
methanolic and water extracts. The methanolic extract
contained trace amounts of components in the ethyl acetate
extract, as observed by TLC. The lyophilized water extract was
processed with methanol to separate fructose and glucose and
thus yielded the proteinaceous fraction K, soluble only in water.
Before purication, extracts and proteinaceous fraction K
were evaluated for their antioxidant and anti-inammatory
activities (Figure 1). We routinely use MTT and LPO assays
to determine the antioxidant activity of natural extracts and
pure isolates. The MTT assay is based on redox reaction and
hence detects most antioxidant compounds that are reducing
agents. On the other hand, inhibition of LPO detects free
radical scavenging capacity of extracts and test compounds.
Biochemical reactions in vivo generate free radicals. The
reaction of free radicals with lipids, proteins, and nucleic acids
result in oxidative damage and leads to a number of diseases
including cancer, cardiovascular disease, and arthritis.
15,18
Antioxidants scavenge these free radicals generated in vivo
and prevent such unwanted biochemical reactions. Similarly,
inammation signaling pathways produce intermediates or
inammation-causing hormones. Cyclooxygenase enzymes,
COX-1 and -2, play a signicant role in producing such inter-
mediates as prostaglandins and thromboxanes. Compounds in
food have the ability to inhibit COX enzymes and hence
prevent or modulate the inammation signaling pathways.
Therefore, the anti-inammatory activity of extracts and isolates
Figure 2. Structures of pure isolates from Ajwa dates: avonoid glycoside (1), triterpenoids (2 and 3), triglyceride (4), steroids (57), mixture of
fatty acids (FA), phthalates (bis(2-ethylhexyl) terephthalate (PH1) and bis(2-ethylheptyl) phthalate) (PH2), and isomers of fructose and glucose.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401371v | J. Agric. Food Chem. 2013, 61, 58345840 5837
of Ajwa dates were tested by measuring the inhibition of COX-1
and -2 enzymes. This assay determines the ability of COX enzymes
to convert arachidonic acid to prostaglandins, which initialize the
inammatory process in the body.
15,18
At 250 g/mL, the ethyl
acetate, methanolic, and water extracts gave absorbance values
of 0.18, 0.25, and 0.37 at 570 nm, respectively (Figure 1A).
Fraction K was the most active and showed an absorbance
value of 0.41 at 50 g/mL, similar to the activity of vitamin C
and TBHQ at 25 g/mL (Figure 1A). The ethyl acetate,
methanolic, and water extracts of Ajwa fruits inhibited LPO by
88, 70, and 91% at 250 g/mL, respectively (Figure 1B). As in
the case of MTT assay, fraction K was the most active among
Figure 3. (A) Absorbance values at 570 nm of compounds 13 at 100 g/mL and compound 4, mixture of fatty acids (FA), bis(2-ethylhexyl)
terephthalate (PH1), and bis(2-ethylheptyl) phthalate (PH2) at 25 g/mL obtained after reaction with MTT at 37 C. Vitamin C and TBHQ were
used as positive controls at 25 g/mL. (B) Inhibition of LPO by compounds 13 at 100 g/mL and compound 4, mixture of fatty acids (FA), bis(2-
ethylhexyl) terephthalate (PH1), and bis(2-ethylheptyl) phthalate (PH2) at 25 g/mL. Commercial antioxidants BHA, BHT, and TBHQ were
tested at 10 M. The oxidation of lipid was initiated by the addition of Fe
2+
ions. (C) COX-1 and COX-2 enzyme inhibitory activities of compounds
14, mixture of fatty acids (FA), bis(2-ethylhexyl) terephthalate (PH1), and bis(2-ethylheptyl) phthalate (PH2) at 50 g/mL concentration and
commercial NSAIDs aspirin, Celebrex, naproxen, and ibuprofen used as positive control at 108, 1, 15, and 12 g/mL, respectively. The various
concentrations of positive controls used in these assays were to yield comparable activity proles between 0 and 100% by test extracts, fraction, and
positive controls alike. The standard error of the mean was represented for n = 4 (P < 0.05, t test, paired, two tailed). For the COX assay, vertical
bars represent the standard deviation of each data point (n = 2).
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf401371v | J. Agric. Food Chem. 2013, 61, 58345840 5838
the extracts tested and showed LPO inhibitory activity by about
100% at 50 g/mL. In the COX inhibitory assays at 100 g/mL,
these extracts showed a higher COX-2 enzyme inhibition, by
59, 48, and 45%, respectively, when compared to the COX-1
enzyme (30, 31, and 32%, respectively) (Figure 1C). However,
the trend was opposite in the case of fraction K.
Purication of extracts, as described under Materials and
Methods, aorded pure isolates chrysoeriol-7-O-(2,6-dirham-
nosyl)-glucoside (1),
20
lup-20(29)-en-3-one (2),
21,22
lupeol (3),
23
1,2-dilinoleoyl-3-stearin (4),
24
-sitosteryl-3-glucopyranoside-
6-O-palmitate (5),
25
-sitosteryl-3-O--glucoside (6),
26,27
-sitosterol (7)
26,27
bis(2-ethylhexyl) terephthalate,
28
and
bis(2-ethylheptyl) phthalate,
29
which were elucidated by
NMR spectroscopic analyses (see Figure 2 for structures).
The fatty acid mixture isolated, contained linoleic, oleic, and
stearic acids.
17,30
The sugars puried from methanolic and
water extracts were monosaccharides and their structures
conrmed as mixtures of -D- and -D-glucopyranose,
31
and
mixtures of -D-fructopyranose and -D-fructofuranose
31
by
proton and carbon NMR spectral experiments. Proton and
carbon NMR spectral data revealed that fraction K was
proteinaceous in nature and hence was not studied further to
elucidate its structure(s). It is important to note that several
avonoid glycosides were detected in the methanolic extract by
analytical TLC. However, the paucity of the extract containing
these phenolics allowed only the isolation and characterization
of compound 1.
The isolates from Ajwa dates were tested for antioxidant and
anti-inammatory activities, as in the case of extracts, using
MTT, LPO, and COX-1 and -2 enzyme inhibitory assays. Com-
pounds 57 were not assayed because their activities have been
reported from our laboratory earlier.
26,32
At 100 g/mL con-
centration, compounds 13 gave absorbance values of 0.28,
0.19, and 0.19, respectively (Figure 3A). Compound 4, the
mixture of fatty acids, and phthalates PH1 and PH2 showed
little or no activity as indicated by the poor absorbance values
of 0.06, 0.07, 0.10, and 0.10 at 25 g/mL concentration. These
compounds were not tested at higher concentrations due to
poor solubility. At 100 g/mL concentration, avonoid glyco-
side 1 showed the highest LPO inhibitory activity at 95%.
Triterpenoids 2 and 3 also showed moderate LPO inhibition at
58 and 66%, respectively, at 100 g/mL concentration. Again,
due to poor solubility compound 4, the mixture of fatty acids,
and phthalates were tested at the highest concentration of
25 g/mL and showed very weak LPO inhibition as indicated
by 23, 25, 13, and 13%, respectively (Figure 3B).
The anti-inammatory activity of the pure isolates from Ajwa
fruits was revealed by their COX-1 and -2 enzyme inhibitions.
At 50 g/mL concentration, compounds 14, the mixture of
fatty acids, and phthalates inhibited COX-1 enzyme by 59, 48,
50, 6, 4, 33, and 26% and COX-2 enzyme by 60, 40, 39, 17, 16,
28, and 27%, respectively (Figure 3C). Among these, avonoid
glycoside 1 showed the highest COX-1 enzyme inhibitory pro-
le, similar to that of aspirin, and COX-2 enzyme inhibition
similar to that of naproxen. Triterpenoids 2 and 3 also showed
moderate COX-1 and -2 enzyme inhibitions, similar to ibuprofen.
This is the rst report of the isolation of compounds 16
from date fruits as well as the active proteinaceous fraction. In
addition, the detailed chemical evaluation and biological
activities of Ajwa fruit and biological activities described for
its pure isolates 14 and the phthalates PH1 and PH2 are
reported for the rst time. Ajwa dates contain 39.06% of
fructose, a major portion of the total sugar in the fruit. It is
important to note that fructose has the lowest glycemic index
among natural sugars and has been proven to be very eective
in controlling glycemia in type-2 diabetic patients.
3335
The
overall composition of Ajwa dates in this study showed that it
contained 13.24% seeds, 6.21% moisture, and 11.01% brous
material. The major metabolites in Ajwa fruit were primary
metabolites, sugars and proteins. Interestingly, only mono-
saccharides were present in Ajwa fruit, composed of isomeric
mixtures of fructose and glucose, totaling about 65% of the total
weight of the fruit. Because fructose was considerably higher
than glucose in its total sugar content, consumption of date
fruits are less harmful to persons having issues with sugar
modulation, as in the case of type-2 diabetics. The presence of
phthalates in date fruits could very well be an artifact from
plastics involved in pre- and postharvest handling of dates from
farms to market. The triglyceride and free fatty acids in date
fruits are probably leachates from its seed because seeds are
the main storage location of fatty acids and their glycerides.
Although we did not fully characterize the proteinaceous frac-
tion, it accounted for 1.33% of the total weight of the fruit. Also,
it showed strong antioxidant and anti-inammatory activities.
This is interesting because secondary metabolites are generally
reported or implied to possess such biological activities. On the
basis of the results presented herein, it is clear that Ajwa date
fruit may have added health benets beyond nutrition and t in
the category of functional foods.

AUTHOR INFORMATION
Corresponding Author
*(M.G.N.) Phone: +1 (517) 355-5191, ext. 1406. Fax: +1
(517) 353-0890. E-mail: nairm@msu.edu.
Notes
The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
This research is a contribution from Michigan State University
AgBioResearch and the College of Food Science and Agriculture,
King Saud University, Riyadh, Saudi Arabia.

ABBREVIATIONS USED
MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide; LPO, lipid peroxidation; COX, cyclooxygenase; TLC,
thin-layer chromatography; UV, ultraviolet; NMR, nuclear
magnetic resonance; TBHQ, tert-butylhydroquinone; BHA,
butylated hydroxyanisole; BHT, butylated hydroxytoluene;
NSAIDs, nonsteroidal anti-inammatory drugs; SLPC, 1-stearoyl-
2-linoleoyl-sn-glycerol-3-phosphocholine; APCI-MS, atmospheric
pressure chemical ionization mass spectrometry; DMSO, dimethyl
sulfoxide

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